Evidence has suggested that cyclic AMP, acting through its specific protein kinases, may regulate mitosis. Biochemical and immunochemical localization of the regulatory subunit of the type II cAMP-dependent protein kinase on microtubules points to mitotic spindle microtubules as at least one possible means of such a regulatory control. In preliminary evidence presented in this proposal, microinjection of inhibitors to the catalytic activity of the cAMP-dependent kinase into mitotic PtK1 cells in culture results in an inhibition of normal mitosis. The mechanism of this inhibition is not known. This proposal outlines a series of experiments designed to further study the possible role of cAMP-dependent protein kinase in the regulation of mitotic events. The experiments address several questions. Are the regulatory and catalytic subunits of the kinase acting together in the mitotic spindle during mitosis? Are the kinase subunits associated with mitotic spindle microtubules, and if so, with any particular subset of microtubules? Can the kinase subunits be localized in association with any other spindle-associated structures, such as the centrosomes or vesicles? Does inhibition of the catalytic activity of the kinase lead to any alteration in spindle morphology which can explain the observed inhibition of spindle function? These questions will be approached in two ways. Polyclonal antibodies directed against the regulatory and catalytic subunits of the cAMP kinase will be produced and characterized for use in immunogold localization of the subunits in mitotic cells in culture. Fluorescently labeled catalytic subunit will also be microinjected into living cells to determine if it becomes associated with any structural components. Finally, mitotic cells inhibited by microinjection of inhibitors to the catalytic activity of the cAMP-dependent kinase will be examined in the electron microscope to determine if any structural alteration can be correlated with the mitotic inhibition.